ArticlePDF Available

Abstract

Sentinel species such as crocodilians are used to monitor the health of ecosystems. However, few studies have documented the presence of zoonotic diseases in wild populations of these reptiles. Herein we analyzed 48 serum samples from Crocodylus acutus (n = 34) and C. moreletii (n = 14) from different sites in the state of Quintana Roo (Mexico) to detect antibodies to Leptospira interrogans by means of a microscopic agglutination test (MAT). Crocodylus acutus and C. moreletii tested positive to 11 and 9 serovars, respectively, with Grippotyphosa being the serovar with the highest prevalence in Cozumel island (100%), Banco Chin- chorro Biosphere Reserve (70.6%), and Rı ́o Hondo (100%), while in Chichankanab Lake, it was Bratislava (75%). Titers ranged from 1:50 to 1:3200, and the most frequent was 1:50 in all study sites. Leptospira is present in fresh and saltwater individuals due to the resistance of the bacterium in both environments. Cases of infected people involved with crocodile handling and egg collection suggest that these reptiles could play an important role in the transmission of leptospirosis. Preventive medicine programs should consider the monitoring of reptiles, and testing the soil and water, to prevent outbreaks of leptospirosis in facilities containing crocodiles.
Evidence for Wild Crocodiles as a Risk for Human
Leptospirosis, Mexico
Jonathan Pe
´rez-Flores ,
1
Pierre Charruau,
2
Rogelio Ceden
˜o-Va
´zquez,
1
and Daniel Atilano
3
1
Departamento de Sistema
´tica y Ecologı
´a Acua
´tica, El Colegio de la Frontera Sur, Unidad Chetumal, Avenida Centenario Km 5.5, C.P. 77014
Chetumal, Quintana Roo, Mexico
2
Centro del Cambio Global y la Sustentabilidad en el Sureste, A.C., Calle Centenario del Instituto Jua
´rez s/n, Col. Reforma, C.P. 86080 Villahermosa,
Tabasco, Mexico
3
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto
´noma de Me
´xico, Circuito Exterior s/n, Ciudad Universitaria C.P. 04510,
Mexico
Abstract: Sentinel species such as crocodilians are used to monitor the health of ecosystems. However, few
studies have documented the presence of zoonotic diseases in wild populations of these reptiles. Herein we
analyzed 48 serum samples from Crocodylus acutus (n= 34) and C. moreletii (n= 14) from different sites in
the state of Quintana Roo (Mexico) to detect antibodies to Leptospira interrogans by means of a microscopic
agglutination test (MAT). Crocodylus acutus and C. moreletii tested positive to 11 and 9 serovars, respectively,
with Grippotyphosa being the serovar with the highest prevalence in Cozumel island (100%), Banco Chin-
chorro Biosphere Reserve (70.6%), and Rı
´o Hondo (100%), while in Chichankanab Lake, it was Bratislava
(75%). Titers ranged from 1:50 to 1:3200, and the most frequent was 1:50 in all study sites. Leptospira is present
in fresh and saltwater individuals due to the resistance of the bacterium in both environments. Cases of infected
people involved with crocodile handling and egg collection suggest that these reptiles could play an important
role in the transmission of leptospirosis. Preventive medicine programs should consider the monitoring of
reptiles, and testing the soil and water, to prevent outbreaks of leptospirosis in facilities containing crocodiles.
Keywords: Zoonosis, Leptospire, Reptiles, Crocodylus acutus,Crocodylus moreletii, Mexican Caribbean
INTRODUCTION
Crocodilians are considered sentinel species in the ecosys-
tems they inhabit (Poletta et al. 2008). Their immune
system is remarkably effective; in vitro studies have
demonstrated that crocodiles have an active antibacterial
system that could allow them to live in septic environments
with high risk of infection (Preecharram et al. 2008). They
live throughout the tropics, where conditions for trans-
mission of diseases such as leptospirosis are favorable
(Bharti et al. 2003). Leptospirosis is the most widespread
zoonosis in the world and has been classified as a re-
emerging infectious disease (Bharti et al. 2003; Aguirre
et al. 2006). Pathogenic leptospires infect susceptible hosts
by direct or indirect contact with body fluids (urine, blood,
and genital secretions) (Heath and Johnson 1994; Acevedo-
Whitehouse et al. 2003; Silva et al. 2009). It has been
documented in terrestrial (Busch 1970; Colegrove et al.
Correspondence to: Jonathan Pe
´rez-Flores, e-mail: johnspf77@yahoo.com.mx
EcoHealth
DOI: 10.1007/s10393-016-1196-7
Original Contribution
Ó2016 International Association for Ecology and Health
2005; Langoni et al. 2009; Moreno-Beas et al. 2015) and
aquatic organisms (Smith et al.1977; Lounsbury et al. 2001;
Colegrove et al. 2005; Kik et al. 2006; Mathews et al. 2012;
Sulzner et al. 2012). Faine et al. (1999) noted that the role
of reptiles in the transmission of pathogenic leptospires is
unknown. Nevertheless, leptospiral agglutinins have been
found in several species of reptiles (Andrews et al. 1965;
Rossetti et al. 2003; Silva et al. 2009; Biscola et al. 2011;
Lindtner-Knific et al. 2013; Rodrigues et al. 2016). The
active infection, based on serology, suggests that amphib-
ians and reptiles may play a role as a reservoir host of some
serotypes of Leptospira spp. (Glosser et al. 1974; Aguirre
et al. 2006).
The American crocodile (Crocodylus acutus) and
Morelet’s crocodile (C. moreletii) are found in Quintana
Roo (QR), Mexico. Crocodylus acutus occurs in coastal
saltwater habitats, and C. moreletii inhabits freshwater
systems (Ceden
˜o-Va
´zquez et al. 2006; Ceden
˜o-Va
´zquez
2011). In recent years, increased coastal development has
caused the fragmentation, destruction, and pollution of the
environment. The reduction of these habitats (swamps,
lakes, lagoons, aguadas, cenotes, and ponds) and their use
by people has increased their exposure to crocodile pa-
thogens. There are few studies reporting infectious diseases
of free-ranging crocodilians (Shotts et al. 1972; Huchzer-
meyer 2003; Rossetti et al. 2003). In QR, Charruau et al.
(2012) reported the presence of 47 species of bacteria in the
oral and cloacal cavities of Morelet’s and American cro-
codiles, which could be potential pathogenic agents for
humans.
Leptospirosis is an important zoonosis in developing
countries. Recent outbreaks in these countries are related
to an increase in ecotouristic activities (Pappas et al.
2008). In the state of QR, these activities include fishing,
diving, swimming, snorkeling, and recently, underwater
encounters with crocodiles and manatees, so these activi-
ties could increase the risk of transmission of Leptospira.
Recently, Sa
´nchez-Montes et al. (2015) reported 16 cases
(including 2 fatal) of leptospirosis in humans in QR
during the period 2000–2010 without mentioning the
source of infection. Feuer and Domash-Martinez (2011)
reported nine cases of leptospirosis in people working with
wild alligators in Florida; therefore, it is necessary to study
the ecology of this bacterium and its ectothermic hosts.
This study determines the seroprevalence of eleven ser-
ovars of Leptospira interrogans in wild American and
Morelet’s crocodiles in QR.
MATERIALS AND METHODS
Study Sites
Samples were collected from American crocodiles captured
on Cozumel island (CI) and Banco Chinchorro Biosphere
Reserve (BCBR), and from Morelet’s crocodiles captured in
´o Hondo (RH) and Chichankanab Lake (CL), QR,
Mexico (Figure 1). In Cozumel island, crocodiles were
captured in the coastal lagoons of Punta Sur Ecological
Park (20°1703900N, 86°5904000W). These shallow lagoons
(±1 m mean depth) are separated from the sea by a narrow
sandy dune and present relatively high salinity during the
dry season (mean >50 ppt in May; Charruau 2010).
Banco Chinchorro is an atoll situated 31 km off the
southern coast of QR. Crocodiles were captured in the
shallow lagoons of Cayo Centro (18°3500400N,
87°1901100W), the largest island of the atoll. These lagoons
also present high salinity rates (>50 ppt, Charruau et al.
2005). The Rı
´o Hondo is a transboundary river between
Mexico and Belize, and lies on 120 km between the town of
La Unio
´n (17°5304200N, 88°5202400W) and the city of
Chetumal (18°2901600N, 88°1900400W). Chichankanab Lake
(19°5204000N, 88°4600400W) has a length of 20 km and a
maximum width of 600 m; this lake lies over a geological
fault and consists of various bodies of water connected
during the rainy season (Covich and Stuiver 1974). The RH
and CL are freshwater systems; during our surveys salinities
were 0.69 and 1.69 ppt, respectively.
Samples Collection
Crocodiles were captured at night using the break-away
snare technique, during spotlight counts as part of ongoing
projects. Samples from each species were collected at dif-
ferent times from each survey location: Crocodylus acutus
from CI (May 2008, May 2009 and September 2011) and
BCBR (April, August and September 2008, and August
2011), and C. moreletii from RH (June 2009) and CL
(August 2011). Each captured crocodile was physically
examined for signs of disease and sex determination, and
their total length was measured (±0.5 cm) from the tip of
the snout to the tip of the tail. Blood samples were obtained
by venipuncture of the post-occipital sinus of the spinal
vein (Myburgh et al. 2014) using 5 and 10 ml syringes
with a 21 gage (0.8 mm) 95/8’’ (16 mm) needle and
were placed in sterile vacutainers without anticoagulant
Jonathan Pe
´rez-Flores et al.
(Vacutainer, Becton Dickinson and Company Mexico,
Mexico D.F., Mexico). The blood samples were deposited on
wet ice and transported to the laboratory for serum sepa-
ration. In all instances, sera were separated by centrifugation
(3000 G for 15 min) and stored at -20°C until the Micro-
scopic agglutination tests (MAT) were performed.
Microscopic Agglutination Test (MAT)
Serum samples were sent to the certified Laboratory of the
Department of Microbiology and Immunology of the
Universidad Nacional Auto
´noma de Me
´xico. Standard
serologic microscopic agglutination tests (MAT) were
performed as described by Myers (1985). We used 12 live
strains of L. interrogans as antigens: Autumnalis, Bataviae,
Bratislava, Canicola, Celledoni, Grippotyphosa, Hard-
joprajitno, Icterohaemorrhagiae, Pomona, Pyrogenes, Tar-
assovi, and Wolffi. Samples were diluted 1:50 (1 mL of
physiological saline and 25 lL of serum) for screening. We
placed 50 lL of the dilution and 50 lL aliquots of the
antigen of each serovar into 96-flat-bottom microwell
plates (Nalge Nunc International, Rochester, New York,
USA). Negative control (physiological saline with the
antigen) was included for each serovar tested. Plates were
Figure 1. Location of study areas of American and Morelet’s crocodiles in the state of Quintana Roo, Mexico. Crocodylus acutus:Cozumel
Island and Banco Chinchorro Biosphere Reserve; Crocodylus moreletii:ChichankanabLakeandRı
´oHondo.
Prevalence of Leptospira antibodies in Wild Crocodiles
gently stirred and incubated at 37°C for two hours, and
then read by dark field microscopy using a 10X objective.
Twofold serial dilutions were performed for each serum
sample (1:50 to 1:3200) to determine the titer for each
serovar, while agglutination titers were expressed as the
reciprocal of the maximum dilution, at which grade two
agglutination was observed.
Statistical Analysis
For each species, we tested the relation between the number
of different serovar antibodies in crocodile’s blood and TL
and sex of individuals, using a Spearman correlation test
and a Mann–Whitney test, respectively. The analyses were
performed with the software G-Stat 2.0.1. Results were
considered significant at P0.05.
Literature Review
We used the databases of the digital library of the
Universidad Nacional Auto
´noma de Me
´xico and the
Google search engine to search for any publications refer-
ring the presence of antileptospiral antibodies in wild and
captive reptiles.
Table 1. Number of Crocodylus acutus and C. moreletii testing positive for Leptospira using MAT according to serovar and titers.
Leptospira interrogans Serovar Crocodylus acutus n (titer) Crocodylus moreletii n (titer)
Autumnalis 12 (1/50) 2 (1/100) 2 (1/100)
Bataviae 4 (1/50) 2 (1/100) 2 (1/50) 1 (1/100) 1 (1/200)
Bratislava 12 (1/50) 5 (1/100) 2 (1/200) 7 (1/50) 1 (1/100) 1 (1/200) 1 (1/800)
Canicola 8 (1/50) 2 (1/100) 3 (1/50) 3 (1/100)
Grippotyphosa 7 (1/50) 9 (1/100) 12 (1/200) 1 (1/800) 4 (1/50) 2 (1/100) 2 (1/200) 2 (1/400) 1 (1/3200)
Hardjoprajitno 11 (1/50) 2 (1/100) 1 (1/200) 4 (1/50) 1 (1/100)
Icterohaemorrhagiae 1 (1/50)
Pomona 6 (1/50) 4 (1/100) 1 (1/200) 4 (1/50) 2 (1/100)
Pyrogenes 5 (1/50) 8 (1/100) 2 (1/200) 2 (1/50) 1 (1/100)
Tarassovi 3 (1/50)
Wolffi 1 (1/50) 4 (1/100) 2 (1/400) 7 (1/50)
Table 2. Seroprevalence of crocodiles to 11 Leptospira serovars using MAT.
Serovar Seroprevalence (%)
Crocodylus acutus Crocodylus moreletii
CI (n= 17) BC (n= 17) RH (n=6) CL(n=8)
Autumnalis 47 35.3 16.7 12.5
Bataviae 29.4 5.9 33.3 25
Bratislava 64.7 47 66.7 75
Canicola 23.5 35.3 50 37.5
Grippotyphosa 100 70.6 100 62.5
Hardjoprajitno 58.8 23.5 50 25
Icterohaemorrhagiae 0 5.9 0 0
Pomona 41.2 23.5 83.3 12.5
Pyrogenes 64.7 23.5 33.3 12.5
Tarassovi 11.8 5.9 0 0
Wolffi 23.5 17.6 83.3 25
CI Cozumel Island, BC Banco Chinchorro, RH
´o Hondo, CL Chichankanab Lake.
Jonathan Pe
´rez-Flores et al.
RESULTS
Microscopic Agglutination Tests
We obtained serum samples from 48 crocodiles, from
which 34 were American crocodiles [73–293 cm total
length (TL); 23 males and 11 females] and 14 were More-
let’s crocodiles (102–173 cm TL; seven males and seven
females) (Table 1).
American Crocodile
In CI, samples werepositive to 10 serovars; theserovar with the
highest prevalence was Grippotyphosa (100%) and the lowest
was Tarassovi (11.8%) (Table 2). Four serovars had 50% of
high prevalence (Bratislava, Grippotyphosa, Hardjopra-
jitno, and Pyrogenes). In this site, titers range from 1:50 to
1:400, where 1:50 was the most frequent (59.7%). In BCBR,
samples were positive to 11 serovars, the highest seropreva-
lence was detected for the serovar Grippotyphosa (70.6%),
and the lowest were for Bataviae, Icterohaemorrhagiae, and
Tarassovi (5.9%) (Table 2).In this case, only one serovar had a
higher prevalence >50%. The titers ranged from 1:50 to 1:800,
where the most frequent were 1:50 (47%) and 1:100 (40%).
Morelet’s Crocodile
In both sites, samples were positive to nine serovars; in RH
the serovar with the highest prevalence was Grippotyphosa
Table 3. List of Leptospira serovars reported in snakes.
Species Serovars Reference
Hognosed snake (Heterodon platirhinos) Ballum Ferris et al. (1961)
Common garter snake (Thamnophis sirtalis) Pomona Abdulla and Karstad (1962)
Midland water snake (Natrix sipedon pleuralis) Ballum, Hardjoprajitno Andrews et al. (1965)
Racers (Coluber constrictor) Ballum, Canicola, Hyos, Sejroe Andrews et al. (1965)
Gray rat snake (Elaphe obsoleta spiloides) Ballum, Canicola, Sejroe Andrews et al. (1965)
Prado’s lancehead snake (Bothrops pradoi) Andamana Hyakutake et al. (1980)
South American rattlesnake (Crotalus durissus
terrificus)
Andamana, Cynopteri, Bataviae, Hardjoprajitno,
Icterohaemorrhagiae, Panama, Sentot
Santa Rosa et al. (1980)
Biscola et al. (2011)
Crossed pit viper (Bothrops alternatus) Andamana, Patoc, Pomona, Pyrogenes,
Shermani, Tarassovi, Wolffi
Stanchi et al. (1986)
Venezuelan anaconda (Eunectes murinus) Autumnalis, Bratislava, Copenhageni,
Icterohaemorrhagiae, Kennewick
Calle et al. (2001)
Brazilian lancehead (Bothrops moojeni) Grippotyphosa, Hardjoprajitno Biscola et al. (2011)
Jararaca (Bothrops jararaca) Hardjomini, Hardjoprajitno, Patoc Biscola et al. (2011)
Neuwied’s lancehead (Bothrops pauloensis) Hardjoprajitno, Pyrogenes Biscola et al. (2011)
Whitetail lancehead (Bothrops leucurus) Hardjoprajitno, Biscola et al. (2011)
Amarali Boa (Boa constrictor amarali) Panama Biscola et al. (2011)
Hardwicke’s Rat Snake (Platyceps
ventromaculatus)
Tarassovi Lindtner-Knific et al. (2013)
Brown Sand Boa (Eryx johnii) Copenhageni, Grippotyphosa, Pomona,
Tarassovi
Lindtner-Knific et al. (2013)
Nose-horned (Vipera ammodytes) Copenhageni Lindtner-Knific et al. (2013)
Rough tailed sand boa (Gongylophis conicus) Tarassovi Lindtner-Knific et al. (2013)
Herald snake (Crotaphopeltis hotamboeia) N/I Jobbins and Alexander (2015)
Tropical Rattlesnake (Crotalus durissus
collilineatus)
Australis, Autumnalis, Andamana, Bataviae,
Bratislava, Canicola, Copenhageni, Cynopteri,
Djasiman, Grippotyphosa, Hardjoprajitno,
Hebdomadis, Javanica, Panama, Patoc,
Pomona, Pyrogenes, Sentot, Tarassovi,
Whitcomb, Wolffi
Rodrigues et al. (2016)
Prevalence of Leptospira antibodies in Wild Crocodiles
(100%) and the lowest was Autumnalis (16.7%) (Table 2).
Six serovars had 50% of higher prevalence (Bratislava,
Canicola, Grippotyphosa, Pomona, and Wolffi). Titers
ranged from 1:50 to 1:800, where the most frequent was
1:50 (68%). On the other hand, the serovar with the highest
prevalence in CL was Bratislava (75%) and the lowest were
Autumnalis (12.5%), Pomona (12.5%), and Pyrogenes
(12.5%) (Table 2). Only two serovars had 50% of higher
prevalence (Bratislava and Grippotyphosa). Titers ranged
from 1:50 to 1:3200, where the most frequent was 1:50
(50%).
Analysis
There was no significant relation between TL of C. moreletii
and the number of different Leptospira serovars antibodies
(Rho spearman = 0.4229; t= 1.6165; P= 0.1320). For C.
acutus, there was a significant negative relation between its TL
and number of Leptospira serovars in crocodiles blood (Rho
spearman = -0.4260; t=-2.7041; P= 0.0107). In both
species, the mean number of Leptospira serovars in the blood
of individuals was not different between sexes (C. moreletii:
U= 0.3898, P= 0.6967; C. acutus:U= 0.000; P=1).
Table 4. List of Leptospira serovars reported in turtles.
Species Serovars Reference
European pond turtle (Emys orbicularis) Biflexa, Tarassovi, Copenhageni, Grippoty-
phosa
Combiesco et al. (1964)
Lindtner-Knific et al. (2013)
Snapping turtle (Chelydra serpentina) Hyos, Hardjoprajitno, Ballum Andrews et al. (1965)
Eastern box turtle (Terrapene carolina carolina) Hyos, Ballum, Canicola, Icterohaemorrhagiae,
Pomona
Andrews et al. (1965)
Red eared turtle (Pseudemys scripta elegans) Hyos, Ballum, Hardjoprajitno, Pomona,
Tarassovi
Andrews et al. (1965)
Glosser et al. (1974)
Lindtner-Knific et al. (2013)
Caspian turtle (Mauremys caspica) Biflexa, Ballum, Hyos, Tarassovi van der Hoeden (1968)
D’Orbigny’s slider (Trachemys dorbigni) Andamana, Bataviae, Canicola, Garcia, Grip-
potyphosa, Icterohaemorrhagiae, Tarassovi
Silva et al. (2009)
Hilaire’s toadhead turtle (Phrynops hilarii) Icterohaemorrhagiae Silva et al. (2009)
Spur thighed tortoise (Testudo graeca) Grippotyphosa, Pomona Lindtner-Knific et al. (2013)
Hermann’s tortoise (Testudo hermanni) Grippotyphosa Lindtner-Knific et al. (2013)
Geoffroy’s toadhead turtle (Phrynops geoffroanus) Andamana, Australis, Autumnalis, Bataviae,
Butembo, Canicola, Castellonis, Copen-
hageni, Grippotyphosa, Hebdomadis,
Icterohaemorrhagiae, Patoc, Pomona,
Pyrogenes, Shermani, Sentot, Whitcombi,
Wolffi
Oliveira (2013)
Blanding’s turtle (Emydoidea blandingii) Bratislava, Canicola, Grippotyphosa, Ictero-
haemorrhagiae
Grimm et al. (2015)
Giant Amazon turtle (Podocnemis expansa) Andamana, Australis, Autumnalis, Bataviae,
Bratislava, Butembo, Castellanos, Cynop-
teri, Hardjoprajitno, Hebdomadis, Ictero-
haemorrhagiae, Javanica, Panama, Patoc,
Pomona, Pyrogenes, Sentot, Whitcombi
Alves-Ju
´nior (2013)
Hawksbill sea turtle (Eretmochelys imbricata) Bratislava, Canicola, Grippotyphosa, Hard-
joprajitno, Wolffi
Pe
´rez-Flores unpublished data
Green sea turtle (Chelonia mydas) Autumnalis, Bratislava, Canicola, Celledoni,
Grippotyphosa, Hardjoprajitno, Wolffi
Pe
´rez-Flores unpublished data
Jonathan Pe
´rez-Flores et al.
Literature Review Results
We found 19 studies referring to the presence of antilep-
tospiral agglutinins in reptiles. These investigations re-
ported leptospiral antibodies in 19 species of snakes
(Table 3), 14 of turtles (Table 4), eight lizards (Table 5),
and two crocodilians (Table 6).
DISCUSSION
Recently, crocodilians have been used for different kinds of
activities (petting, educational programs, ranching, farm-
ing, and underwater encounters), and due to their great
antimicrobial resistance, little attention has been given to
the preventive medicine programs in these reptiles. Most of
the pathogens affecting crocodilians have been reported in
captive and farmed animals (Huchzermeyer 2003), and few
studies have documented the presence of zoonotic patho-
gens in wild individuals (Huchzermeyer 2003; Rossetti et al.
2003; Charruau et al. 2012). Probably the most reported
zoonotic disease in reptiles is salmonellosis (Mermin et al.
2004), but diseases like leptospirosis have been underesti-
mated and neglected as a disease that could be transmitted
by reptiles (Faine et al. 1999). Feuer and Domash-Martinez
(2011) and Snelling et al. (2004) reported cases of lep-
tospirosis in people involved with crocodilian handling
activities. In spite of the potential risks, as far as we know,
only one study detected leptospiral antibodies in wild and
captive crocodilians (Rossetti et al. 2003). To our knowl-
edge, this is the first study investigating the presence of
serum antileptospiral agglutinins in both C. acutus and C.
moreletii.
This study shows high antibodies prevalence of seven
serovars (50%) in all sites sampled. In CI and BCBR,
American crocodiles tested positive to 10 and 11 serovars,
respectively; but in the case of BCBR, only one serovar
(Grippotyphosa) had 50% of prevalence (Table 2), per-
haps because it is a natural protected area and several
programs have been applied to eradicate invasive species
(cats, rats, and mice). Most of the serovars detected in
crocodiles of CI have been reported previously in species
that could be part of their diet: Autumnalis and Canicola
were reported in invasive (Mus musculus and Rattus rattus),
and endemic (Oryzomys couesi cozumelae) rodents (So-
tomayor 2009); Autumnalis, Bratislava, and Canicola were
reported as common serovars in the pygmy raccoon (Pro-
cyon pygmaeus); Autumnalis, Bratislava, Canicola, and
Pyrogenes in dogs of urban and rural areas of CI (Mena
2007).
The site with the highest number of serovars with
50% of prevalence was RH (Table 2). The poor sanitary
measures and the introduction of potential reservoirs could
have increased the transmission of leptospirosis in this area.
During the surveys, we observed the presence of domestic
(i.e., dogs, pigs, and cattle) and wild animals, as well as
invasive rodents. Four of the serovars (Canicola, Grippo-
typhosa, Hardjoprajitno, and Pomona) detected in croco-
diles of RH were previously reported in cattle sampled in
villages near the zone (Pen
˜a1987). Additionally, in QR, the
Antillean manatee (Trichechus manatus manatus) another
sentinel species that moves throughout the RH has been
reported with antibodies against five serovars (Bratislava,
Canicola, Grippotyphosa, Hardjoprajitno, and Pomona)
(Sanvicente-Lo
´pez 2005), which were also found in the
crocodiles of RH analyzed in this study (Table 2).
Chichankanab Lake presented the lowest number of
serovars with 50% of prevalence. This site has been used
as recreational area, and apparently no agricultural activi-
Table 5. List of Leptospira serovars reported in lizards.
Species Serovars Reference
Sand lizard (Lacerta agilis) Sejroe Plesko et al. (1964)
Hardwick’s spiny tailed Lizard (Uromastyx hardwickii) Copenhageni, Grippotyphosa, Tarassovi Lindtner-Knific et al. (2013)
Sudan spiny tailed lizard (Uromastyx dispar) Australis, Grippotyphosa, Tarassovi Lindtner-Knific et al. (2013)
Leopard gecko (Eublepharis macularius) Australis, Canicola, Grippotyphosa, Tarassovi Lindtner-Knific et al. (2013)
Green iguana (Iguana iguana) Hardjoprajitno, Tarassovi Lindtner-Knific et al. (2013)
Green basilisks (Basiliscus plumifrons) Grippotyphosa Lindtner-Knific et al. (2013)
Black agama lizard (Laudakia melanura) Grippotyphosa Lindtner-Knific et al. (2013)
Common wonder gecko (Teratoscincus scincus) Grippotyphosa Lindtner-Knific et al. (2013)
Prevalence of Leptospira antibodies in Wild Crocodiles
ties take place around the lake. The only potential reser-
voirs in this site are wildlife, invasive rodents, and rural
dogs. In the Yucata
´n Peninsula, dogs have been associated
with the serovars Grippotyphosa, Pomona, Pyrogenes, and
Canicola; rodents with Wolffi and Bratislava; and seven
(Bratislava, Canicola, Hardjoprajitno, Pomona, Grippoty-
phosa, Pyrogenes, and Wolffi) of the nine serovars reported
in humans (Vado-Solı
´s et al. 2002; Jimenez-Coello et al.
2008) were found in crocodiles captured in CL (Table 2).
Apparently, the sex of individuals does not affect the
possibility of infection by Leptospira in both crocodile
species; in C. moreletii, the size (i.e., age) of crocodiles
apparently does not affect the possibility of infection. On
the contrary, in C. acutus, our result shows that the number
of different Leptospira serovars antibodies in the blood of
crocodiles decreases significantly with the increase of cro-
codiles size. However, the sample size used in this study is
too small and there is not enough evidence to suggest that
these results are definitive. The significant relation found in
C. acutus could be due to differences in habitat use between
size classes. Crocodile populations of Banco Chinchorro
atoll and Cozumel Island are both relatively dense (Char-
ruau et al. 2005; Gonza
´lez-Corte
´s2007), which promotes
intraspecific competition. Usually, in situations of territo-
rial conflicts, smaller individuals are less competitive and
are steered to lower quality sites by larger crocodiles
(Thorbjarnarson 1989). Thus, juveniles and sub-adults are
often found in marginal habitats such as hypersaline water
or exposed shorelines (Thorbjarnarson 1989). Thus, we
suggest that lower quality sites would favor the survival
and/or proliferation of Leptospira, increasing the infection
risks for smaller crocodiles evolving in those habitats. Fu-
ture researches should study the presence and abundance of
Leptospira in different types of habitats used by crocodiles
to validate this hypothesis.
One of the principal routes of infection of leptospirosis
is via contaminated soil or water (Bharti et al. 2003). The
presence of antibody titers in crocodiles inhabiting fresh
and salt waters could be related to the ability of the bac-
terium to survive in both scenarios. Saito et al. (2004)
stated that Leptospira could survive three days in seawater;
and Andre-Fontaine et al. (2015) documented that it can
survive for at least 20 months in freshwater. Additionally,
the behavior of American and Morelet’s crocodiles can play
an important role in the transmission of the disease because
they spend time basking along the shore of bodies of water
(nesting, hunting, eating, and resting), where the water
usually is shallow and the temperature is higher. These
conditions create a favorable environment for the trans-
mission of this zoonotic disease. In the Yucata
´n Peninsula,
cases of leptospirosis in humans have been reported since
1920, where this disease usually has affected children and
young adults, and has been linked to occupational and
recreational activities (Sakata et al. 1992; Vado-Solı
´s et al.
2002). In all sites sampled in this study, people conduct
activities involving contact with water, soil, and food that
could be contaminated with animal urine. Many of the
ecotouristic recreational centers in QR are lagoons, cenotes,
aguadas, lakes, rivers, and swamps inhabited by crocodiles.
Zavala-Vela
´zquez et al. (2008) reported three cases of lep-
tospirosis in people that swam in sinkholes and lagoons. In
two cases, the serological tests were positive to the serovar
Grippotyphosa, the serovar with the highest seroprevalence
found in this study. In the other case, the individual was
positive to the serovar Canicola, which was also detected in
these crocodiles.
Several studies have suggested that the principal source
of transmission of leptospirosis are domestic and wild
mammals (Levett 2001; Bharti et al. 2003), but the cases of
infected people involved with crocodile handling and egg
Table 6. List of Leptospira serovars reported in crocodilians.
Species Serovars Reference
Yacare caiman (Caiman yacare) Copenhageni, Javanica, Pyrogenes, Sarmin Rossetti et al. (2003)
Broad-snouted caiman (Caiman latirostris) Copenhageni, Javanica, Pyrogenes, Sarmin Rossetti et al. (2003)
American crocodile (Crocodylus acutus) Autumnalis, Bataviae, Bratislava, Canicola, Grippotyphosa,
Hardjoprajitno, Icterohaemorrhagiae, Pomona,
Pyrogenes, Tarassovi, Wolffi
This study
Morelet’s crocodile (Crocodylus moreletii) Autumnalis, Bataviae, Bratislava, Canicola, Grippotyphosa,
Hardjoprajitno, Pomona, Pyrogenes, Wolffi
This study
Jonathan Pe
´rez-Flores et al.
collection reported by Feuer and Domash-Martinez (2011)
and Snelling et al. (2004) suggest that these activities could
be extremely risky. The serovar Hardjoprajitno, reported in
two crocodile handlers in Australia, was also found in 19
crocodiles in this study.
Apparently, reptiles do not present clinical signs of
leptospirosis despite the fact that in some species titers of
1600 (Rodrigues et al. 2016), 3200 (Biscola et al. 2011),
and 6400 (Hyakutake et al. 1980) have been detected.
However, in this study, the individual with the highest
titers (1:3200) presented a low body condition, and a
lower weight compared with other crocodiles of the same
size. Huchzermeyer (2003) indicated that bacterium pre-
sent in the environment and in food could cause sep-
ticemias in crocodiles. These bacteremias are the result of
an immunosuppression originated by continuous stressful
episodes. If these stressors are not removed, animals could
die due to stress-associated disease (Huchzermeyer 2003).
For these reasons, we need to perform routine examina-
tions in captive and farmed crocodilians; MAT tests
should be conducted when animals are quarantined to
prevent the transmission of this zoonosis. The staff in-
volved in the management of crocodiles in zoos, farms,
and in the wild must take precautions to minimize the
risk of exposure. In addition, all institutions holding
confined crocodilians need to develop a monitoring pro-
gram to detect the presence of leptospirosis in soil or
water.
CONCLUSIONS
This study demonstrates that all (100%) of the fresh and
saltwater crocodiles sampled present antileptospiral agglu-
tinins. The resistance of the bacterium to both environ-
ments increases the risk of transmission of this zoonotic
disease, and thus, precautions should be taken when
recreational activities are carried out in places inhabited by
crocodiles. Serovars Hardjoprajitno and Australis have
been associated in cases of infected people working with
reptiles: for this reason, control and preventive measures
must be taken by personnel handling captive and wild
reptiles. Leptospirosis has been underestimated and mis-
diagnosed due to the absence of pathognomonic signs. For
its part, the MAT test has proved to be a useful technique
for detecting cases of leptospirosis in ectothermic and
endothermic hosts.
ACKNOWLEDGEMENTS
Our most sincere thanks to H. Gonza
´lez-Corte
´s, J. Pe
´rez-
Jua
´rez, F. Gonza
´lez-A
´vila, C. Tuz-Catzin, R. Rosas-Car-
mona, C. Lo
´pez-Miam, and O. Martı
´nez-Castillo for field
assistance. JPF thanks R. Ibarra for helping in the
manuscript. Special thanks to the Fundacio
´n de Parques y
Museos de Cozumel and to Banco Chinchorro Biosphere
Reserve (CONANP). We thank the Secretarı
´a de Marina
(SEMAR) for the logistic support during the surveys
conducted along the Rı
´o Hondo. The Secretarı
´ade
Educacio
´nPu
´blica (SEP) and the Comisio
´n Nacional para
el Conocimiento y Uso de la Biodiversidad (CONABIO)
provided financial support. The map was produced by H.
Weissenberger. We also thank L.W. Porras for comments
that improved the manuscript. The Secretaria de Medio
Ambiente y Recursos Naturales (SEMARNAT) of Mexico
provided the scientific research permissions for crocodiles
capture (Oficios Nu
´m. SGPA/DGVS/02066/08, 02516/09,
04528/11, 02082/11).
REFERENCES
Abdulla PK, Karstad L (1962) Experimental infections with Lep-
tospira pomona in snakes and turtles. Zoonoses Research 1:295–
306
Acevedo-Whitehouse K, de la Cueva H, Gulland FM, Aurioles-
Gamboa D, Fausto Arellano-Carbajal F, Suarez-Gu
¨emes F
(2003) Evidence of Leptospira interrogans infection in California
sea lion pups from the gulf of California. Journal of Wildlife
Diseases 391:145–151
Aguirre AA, Gardner SC, Marsh JC, Delgado SG, Limpus CJ,
Nichols WJ (2006) Hazards Associated with the Consumption
of Sea Turtle Meat and Eggs: A Review for health Care Workers
and the General Public. EcoHealth 3:141–153
Alves-Ju
´nior JR (2013) Leptospira spp. e Brucella spp em tartaru-
gas-da-amazonia (Podocnemis expansa) do vale do rio Araguaia-
GO. Ph.D. Thesis. Universidade Estadual Paulista, Brasil
Andre-Fontaine G, Aviat F, Thorin C (2015) Waterborne Lep-
tospirosis: survival and preservation of the virulence of patho-
genic Leptospira spp. in fresh water. Current Microbiology 7:136–
142. doi:10.1007/s00284-015-0836-4
Andrews RD, Reilly JR, Ferris D, Hanson LE (1965) Leptospiral
agglutinins in sera from Southern Illinois Herpetofauna. Journal
of Wildlife Diseases 1:55–59
Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett
MA, et al. (2003) Leptospirosis: a zoonotic disease of global
importance. The Lancet Infectious Diseases 3:757–771
Biscola NP, FornazarI F, Saad F, Richini-Pereira VB, Campagner
MV, Langoni H, et al. (2011) Serological investigation and PCR
in detection of pathogenic leptospires in snakes. Pesquisa Ve-
terina
´ria Brasileira 31:806–811
Prevalence of Leptospira antibodies in Wild Crocodiles
Busch AL (1970) Epizootiology and epidemiology of leptospirosis.
Journal of Wildlife Diseases 6:273–274
Calle PP, Rivas J, Mun
˜oz M, Thorbjarnarson J, Holmstrom W,
Karesh WB (2001) Infectious disease serologic survey in free-
ranging Venezuelan anacondas (Eunectes murinus). Journal of
Zoo and Wildlife Medicine 32:320–323
Ceden
˜o-Va
´zquez JR, Ross JP, Calme
´S (2006) Population status
and distribution of Crocodylus acutus and C. moreletii in
southeastern Quintana Roo, Mexico. Herpetological Natural
History 10:53–66
Ceden
˜o-Va
´zquez JR (2011) El cocodrilo: recurso milenario. In:
Riqueza biolo
´gica de Quintana Roo. Un ana
´lisis para su con-
servacio
´n, Tomo 1, Pozo C, Armijo-Canto N, Calme
´S (editors),
Me
´xico, D.F: El Colegio de la Frontera Sur (ECOSUR), Comi-
sio
´n Nacional para el Conocimiento y Uso de la Biodiversidad
(CONABIO), Gobierno del Estado de Quintana Roo y Pro-
grama de Pequen
˜as Donaciones (PPD), pp 234–240
Charruau P, Ceden
˜o-Va
´zquez JR, Calme
´S (2005) Status and
conservation of the American crocodile (Crocodylus acutus)in
Banco Chinchorro Biosphere Reserve, Quintana Roo, Mexico.
Herpetological Review 36:390–395
Charruau P (2010) Ecologı
´a y etologı
´a de anidacio
´n del cocodrilo
americano (Crocodylus acutus): Un estudio para su conservacio
´n.
Ph.D. Thesis. El Colegio de la Frontera Sur, Me
´xico
Charruau P, Pe
´rez-Flores J, Pe
´rez-Jurez JG, Ceden
˜o-Va
´zquez RJ,
Rosas-Carmona R (2012) Oral and cloacal microflora of wild
crocodiles Crocodylus acutus and C. moreletii in the Mexican
Caribbean. Diseases of Aquatic Organisms 98:27–39
Colegrove MK, Lowenstine LJ, Gulland FM (2005) Leptospirosis
in northern elephant seals (Mirounga angustirostris) stranded
along the California coast. Journal of Wildlife Diseases 41:426–
430
Combiesco D, Sturdza N, Elian M, Nicolesco M (1964) Study on
the animal sources of infection in leptospirosis. Proceedings of
the Second International Symposium on Leptospira an Lep-
tospirosis in Men and Animals, Dublin, Vol 1, pp 249–254
Covich A, Stuiver M (1974) Changes in oxygen 18 as a measure of
long-term fluctuations in tropical lake levels and molluscan
populations. Limnology and Oceanography 19:682–691
Faine S, Adler B, Bolin C, Perolat P (1999) Leptospira and lep-
tospirosis, Melbourne, Australia: MediSci
Ferris DH, Rhoades HE, Hanson LE, Galton MM, Mansfield ME
(1961) Research into nidality of Leptospira ballum in campestral
hosts including the hog-nosed snake (Heterodon platyrhinos).
The Cornell Veterinarian 51:405–419
Feuer B, Domash-Martinez T (2011) Report of case: leptospirosis
after exposure to alligator carcass. Osteopathic Family Physician
3:23–26
Glosser JW, Sulzer CR, Eberhardt M, Winkler WG (1974) Cultural
and serologic evidence of Leptospira interrogans serotype Tar-
assovi infection in turtles. Journal of Wildlife Diseases 10:429–435
Gonza
´lez-Corte
´s H (2007) Estudio poblacional de Crocodylus
acutus (Cuvier, 1807) en el Refugio Estatal de Flora y Fauna
Laguna de Colombia, Cozumel, Quintana Roo, Me
´xico. BSc.
Thesis. Universidad Veracruzana, Me
´xico
Grimm K, Mitchell MA, Thompson D, Maddox C (2015) Sero-
prevalence of Leptospira spp. in Blanding’s Turtles (Emydoidea
blandingii) from DuPage County, Illinois USA. Journal of Her-
petological Medicine and Surgery 25:28–32. doi:10.5818/1529-
9651-25.1.28
Heath SE, Johnson R (1994) Leptospirosis. Journal of the American
Veterinary Medical Association 205:1518–1523
Huchzermeyer FW (2003) Crocodiles: Biology, Husbandry and
Diseases, Wallingford: CABI Publishing
Hyakutake S, Biasi PD, Belluomini HE, Santa Rosa CA (1980)
Leptospiroses in Brazilian snakes. International Journal of Zoo-
noses 7:73–77
Jimenez-Coello M, Vado-Solis I, Ca
´rdenas-Marrufo MF, Ro-
drı
´guez-Buenfil JC, Ortega-Pacheco A (2008) Serological survey
of canine leptospirosis in the tropics of Yucatan Mexico using
two different tests. Acta Tropica 106:22–26. doi:10.1016/j.acta-
tropica.2007.12.011
Jobbins SE, Alexander KA (2015) Evidence of Leptospira sp.
infection among a diversity of African wildlife species: beyond
the usual aspects. Transactions of the Royal Society of Tropical
Medicine and Hygiene 109:349–351. doi:10.1093/trstmh/trv007
KikMJL,GorisMG,BosJH,HartskeerRA,DorresteinGM(2006)
An outbreak of leptospirosis in seals (Phoca vitulina) in captivity.
Veterinary Quarterly 28:33–39. doi:10.1080/01652176.2006.9695204
Langoni H, Kawaguchi MF, Oshika JC, Da Silva RC, Teixeira RC
(2009) Leptospira spp. antibodies in captive coatis (Nasua nasua
Storr, 1780) (Carnivora: Procyonidae). Journal of Venomous
Animals and Toxins Including Tropical Diseases 15:762–767.
doi:10.1590/S1678-91992009000400013
Levett PN (2001) Leptospirosis. Clinical Microbiology Reviews
14:296–326
Lindtner-Knific R, Vergles-Rataj A, Vlahovic K, Zrimsek P, Dovic
A (2013) Prevalence of antibodies against Leptospira sp. in
snakes, lizards and turtles in Slovenia. Acta Veterinaria Scan-
dinavica 55:65–69. doi:10.1186/1751-0147-55-65
Lounsbury VJ, Geracy JR, Yates NS, Arnold J (2001) Serological
evidence of leptospirosis in Florida manatees. In: Proceedings of
the 14th Biennial Conference on the Biology of Marine Mammals,
Vancouver, Canada: Society for Marine Mammalogy
Mathews PD, da Silva VMF, Fernando CW, Rosas CW, d’Affon-
seca NJA, Lazzarini SM, et al. (2012) Occurrence of antibodies
to Toxoplasma gondii and Leptospira spp. in manatees (Triche-
chus inunguis) of the Brazilian Amazon. Journal of Zoo and
Wildlife Medicine 43:85–88. doi:10.1638/2011-0178.1
Mena H (2007) Presencia de Leptospira spp. y moquillo canino en
poblaciones de perros y carnı
´voros silvestres en la Isla Cozumel.
M.Sc. Thesis. Universidad Nacional Auto
´noma de Me
´xico,
Me
´xico
Mermin J, Hutwagner L, Vugia D, Shallow S, Daily P, Bender J,
et al. (2004) Reptiles, amphibians, and human Salmonella
infection: a population-based, case-control study. Clinical
Infectious Diseases 38:253–261
Moreno-Beas E, Abalos P, Hidalgo-Hermoso E (2015) Sero-
prevalence of nine Leptospira interrogans serovars in wild car-
nivores, ungulates and primates from a zoo population in a
metropolitan region of Chile. Journal of Zoo and Wildlife
Medicine 46:774–778. doi:10.1638/2014-0139.1
Myburgh JG, Kirberger RM, Steyl JC, Soley JT, Booyse DG,
Huchzermeyer FW, et al. (2014) The post-occipital spinal ve-
nous sinus of the Nile crocodile (Crocodylus niloticus): its
anatomy and use for blood sample collection and intravenous
infusions. Journal of South African Veterinary Association 85:1–
10. doi:10.4102/jsava.v85i1.965
Myers DM (1985) Manual de me
´todos para el diagno
´stico de
laboratorio de la leptospirosis, Nota Te
´cnica No. 30, Buenos
Aires, Argentina: Centro Panamericano de Zoonosis, OPS/OMS
Oliveira JP (2013) Detecc¸ao de Salmonella spp. e Leptospira spp. em
Phrynops geoffroanus (Ca
´gado-de-barbicha) em ambiente urbano.
M.Sc. Thesis. Universidade Estadual Paulista, Brasil
Jonathan Pe
´rez-Flores et al.
Pappas G, Papadimitriou P, Siozopoulou V, Christou L, Akritidis
N (2008) The globalization of leptospirosis: worldwide inci-
dence trends. International Journal of Infectious Diseases 12:351–
357. doi:10.1016/j.ijid.2007.09.011
Pen
˜a HM (1987) Prevalencia de Leptospirosis bovina en el
municipio de Othon P. Blanco, Quintana Roo. BSc. Thesis.
Universidad Veracruzana, Me
´xico
Plesko I, Janovicova E, Lac J (1964) Contribution to the impor-
tance of coldblooded animals for the circulation of leptospira in
nature. Zentralblatt Bakteriologie 192:482–484
Poletta GL, Larriera A, Kleinsorge E, Mudry MD (2008) Caiman
latirostris (broad-snouted caiman as a sentinel organism for
genotoxic monitoring: Basal values determination of micronu-
cleus and comet assay. Mutation Research 650:202–209
Preecharram S, Daduang S, Bunyatratchata W, Araki T, Tham-
masirirak S (2008) Antibacterial activity from Siamese crocodile
(Crocodylus siamensis) serum. African Journal of Biotechnology
7:3121–3128. doi:10.5897/AJB08.316
Rodrigues TCS, Santos ALQ, Lima AMC, Gomes DO, Brites VLC
(2016) Anti-Leptospira spp.antibodies in Crotalus durissus
collilineatus kept in captivity and its zoonotic relevance. Acta
Tropica 158:39–42. doi:10.1016/j.actatropica.2016.02.006
Rossetti CA, Uhart M, Romero GN, Prado W (2003) Detection of
leptospiral antibodies in caimans from the Argentinian Chaco.
Veterinary Record 153:632–633
Saito M, Miyahara S, Villanueva S, Aramaki N, Ikejiri M, Ko-
bayashi Y, et al. (2004) PCR and culture identification of pa-
thogenic Leptospira spp. from coastal soil in Leyte, Philippines,
after a storm during Super Typhoon Haiyan (Yolanda). Applied
and Environmental Microbiology 80:6926–6932
Sakata EE, Yasuda PH, Romero EC, Silva MV, Lomar AV (1992)
Serovares de Leptospira interrogans isolados de casos de lep-
tospirose humana em Sao Paulo, Brasil. Revista do Instituto de
Medicina Tropical de Sao Paulo 34:217–221
Sa
´nchez-Montes S, Espinosa-Martı
´nez DV, Rı
´os-Mun
˜oz CA,
Berzunza-Cruz M, Becker I (2015) Leptospirosis in Mexico:
Epidemiology and Potential Distribution of Human Cases. PLoS
One 10:e0133720
Santa Rosa CA, Hyakutake S, Pde Biasi, Belluomini HE, Kawar-
abayashi M, Godano A (1980) Epidemiological survey of lep-
tospirosis among snakes in Brazil. II. Crotalus durissus
terrificus. Revista do Instituto Adolfo Lutz 40:9–13
Sanvicente-Lo
´pez M (2005) Diagno
´stico de agentes parasitarios
en el manatı
´del Caribe (Trichechus manatus) en la regio
´del
sureste de Me
´xico. M.Sc. Thesis. El Colegio de la Frontera Sur,
Me
´xico
Shotts EB EB Jr, Gaines JL Jr, Martin L Jr, Prestwood AK (1972)
Aeromonas-induced deaths among fish and reptiles in an eu-
trophic inland lake. Journal of the American Veterinary Medical
Association 161:603–607
Silva EF, Seyffert N, Cerqueira GM, Leihs KP, Athanazio DA,
Valente AL, et al. (2009) Serum antileptospiral agglutinins in
freshwater turtles from southern Brazil. Brazilian Journal of
Microbiology 40:227–230
Smith AW, Brown RJ, Skilling DE, Bray HL, Keyes MC (1977)
Naturally occurring leptospirosis in northern fur seals (Cal-
lorhinus ursinus). Journal of Wildlife Diseases 13:144–148
Snelling T, Krause V, Dempsey K, Symonds M, Dohnt M, Smythe
L, et al. (2004) Leptospirosis in the Top End of the Northern
Territory: an investigation into the occupational risk to crocodile
handlers. The Northern Territory Disease Control Bulletin 11:1–6
Sotomayor JJ (2009) Asociacio
´n de Leptospira y los ratones
ende
´micos y exo
´ticos en la Isla Cozumel, Me
´xico. BSc. Thesis.
Universidad Nacional Auto
´noma de Me
´xico, Me
´xico
Stanchi NO, Grisolı
´a CS, Martino PE, Peluso FO (1986) Presence
of antileptospira antibodies in ophidia in Argentina. Revista
Argentina de Microbiologı
´a18:127–130
Sulzner K, Johnson CK, Bonde RK, Auil-Gomez N, Powell J,
Nielsen K, et al. (2012) Health assessment and seroepidemio-
logic survey of potential pathogens in wild Antillean manatees
(Trichechus manatus manatus). PloS One 7:1–11. doi:10.371/
journal.pone.0044517
Thorbjarnarson JB (1989) Ecology of the American crocodile
(Crocodylus acutus). In: Crocodiles: Their ecology, management,
and conservation, Hall PM (editor), Gland, Switzerland: IUCN-
The World Conservation Union Publications, pp 228–258
Vado-Solı
´sI,Ca
´rdenaz-Marrufo MF, Jime
´nez-Delgadillo B, Alz-
ina-Lo
´pez A, Laviada-Molina H, Suarez-Solı
´s V, et al. (2002)
Clinical epidemiological study of leptospirosis in humans and
reservoirs in Yucatan, Mexico. Revista do Instituto de Medicina
Tropical de Sao Paulo 44:335–340
van der Hoeden J (1968) Agglutination of leptospirae in sera of
fresh water turtles. Antonie van Leeuwenhoek 34:458–464
Zavala-Vela
´zquez J, Ca
´rdenas-Marrufo M, Vado-Solı
´s I, Cetina-
Ca
´mara M, Cano-Tur J, Laviada-Molina H (2008) Hemorrhagic
pulmonary leptospirosis: three cases from the Yucatan penin-
sula, Mexico. Revista da Sociedade Brasileira de Medicina
Tropical 41:404–408
Prevalence of Leptospira antibodies in Wild Crocodiles
... Several animals (wild and domestic), as well as accidentally humans, are involved in the leptospirosis infection cycle (Torres-Castro et al., 2018). The role of reptiles in the transmission of pathogenic leptospires is unknown (Faine et al., 1999), however antibodies to leptospira have been found in several reptile species (Rossetti et al., 2003;Oliveira et al., 2016;Rodrigues et al., 2016;P erez-Flores et al., 2017;Paz et al., 2019). Caiman latirostris inhabits large wetlands, which are home of a rich diversity of fauna (Larriera and Imhof, 2006), and which provide appropriate conditions for the transmission of this disease. ...
... The serogroup with the highest titer was Pyrogenes, and this same serogroup was registered by Pereira de Olivera (2014) in Brazil, Rossetti et al. (2003) in Chaco Province, andP erez-Flores et al. (2017) in Mexico. This would confirm a wide distribution of this serogroup, found in tropical and subtropical climates. ...
... This was expected due to high densities and temperatures involved with intensive rearing facilities. If we make a comparison with the work done by P erez- Flores et al. (2017), in which they use the same cut-off titer (1:50), the seroprevalence observed in our wild animals is lower than the results they reported. In the case of the work carried out by Paz et al. (2019), the titer considered was 1:100 obtaining 95.6% of animals in captivity with antibodies to leptospira, which would also mean that the seroprevalence found in our study (74%) is lower. ...
Article
Full-text available
Leptospirosis is a disease caused by pathogenic spirochetes of the genus Leptospira, transmitted by wild and domestic animals. Rodents play a fundamental role in the transmission cycle of this zoonosis but the function of reptiles is unknown. For example, crocodilians could play an important role in the transmission of this disease by living in ideal environments (bodies of shallow water and high temperatures) for the colonization of this bacterium. However, few studies have documented the presence of zoonotic diseases in caiman populations. Our objective was to assess the prevalence of antibodies to leptospira and the presence of Leptospira spp. in wild and captive Caiman latirostris. Blood samples were taken from 45 individuals (20 wild and 25 captive). Before extraction, we cleaned each caiman's neck in order to prevent contamination of samples. We determined the presence of antibodies in serum by microscopic agglutination test (MAT) and polymerase chain reaction (PCR) to detect DNA of the bacteria. We excluded 9 of the 45 samples analyzed by MAT because 5 had lipemic serum and 4 were contaminated (colonized by other organisms). Of the 36 caimans studied by microscopic agglutination test (MAT), 56% (20/36) were considered reactive (titers ≥50). In 74% (14/19) of captive samples and 35% (6/17) of wild samples, antibodies to leptospira were detected by MAT. The serogroup with highest occurrence was Pyrogenes (85%, n = 17/20), presenting coagglutinations with Icterohaemorrhagiae (25%, n = 5/20). One sample from a captive animal was positive for PCR, and we could not isolate leptospires because of agar contamination. Of the 45 blood agar media, 17.8% were contaminated and the rest were negative. This work determined the presence of Leptospira spp. in one caiman and a high prevalence of antibodies in captive caiman relative to wild individuals.
... Grippotyphosa resulted the serovar with the highest prevalence with antibody titers between 1:50 and 1:3 200, followed by Pomona, Wolfii and Bratislva. Even though crocodiles are not strictly in contact with humans, cases of infected people involved with handling and egg collection suggest that these cold-blooded animals could play an important role in the transmission of leptospirosis in some environments [59] . ...
... Only Perez-Flores et al. observed that crocodiles with the highest titers presented a generally bad condition and a lower weight than crocodiles of the same size. Septicemia due to leptospirae in crocodiles has been supposed when the animals are under continuous stress conditions [59] . However, it can be stated that reptiles may be involved in the epidemiology of leptospirosis, without developing disease. ...
Article
Full-text available
Captive reptiles, always more often present in domestic environment as pets, may harbor and excrete a large variety of zoonotic pathogens. Among them, Salmonella is the most well-known agent, whereas there are very scant data about infections by mycobacteria, chlamydiae and leptospirae in cold-blooded animals. However, the investigations that found antibody reactions and/or the bacteria in samples collected from free-ranging and captive reptiles show that herpetofauna may be involved in the epidemiology of these infections. The present review reports the updated knowledge about salmonellosis, mycobacteriosis, chlamydiosis and leptospirosis in reptiles and underlines the risk of infection to which people, mainly children, are exposed.
... Some individuals have been recorded by camera trap passing over crocodile nests (Fig. 2D), but they never preyed on eggs (Charruau and Hénaut 2012). However, they could be the cause of the presence of Leptospira interrogans on the atoll (Pérez-Flores et al. 2017). A first assessment of the rat population on Cayo Centro was conducted in 2003, suggesting a small population restricted to the highest part of the island, near human constructions (Charruau 2003). ...
Article
Full-text available
Banco Chinchorro Atoll, the largest atoll in the Caribbean and Mexico, was declared a biosphere reserve due to its cultural, economic, and biological importance. The diversity of fish, birds, and reptiles in its aquatic and terrestrial environments has been well studied. However, knowledge about its richness in mammals is scarce and the existing information has not been synthesized to date. The objective of this work is therefore to search and review the existing literature on the mammals of Banco Chinchorro, and to present a first synthesis on the mastofauna of the atoll. Thirty documents mentioning mammals at Banco Chinchorro were found, from which eight species were identified. Of these, three species were invasive and have been eradicated, one native species became extinct, and four native species still occur in the reserve, two bats and two cetaceans. With these four mammal species, the number of known vertebrates at Banco Chinchorro is now 360. More research on the mammals of Banco Chinchorro is needed to increase the knowledge of the ecology and the population status of the species present in the area. It is also very likely that other species of bats and cetaceans are present in the reserve.
... Despite this, hatchlings should not be touched by the public due to the possibility of zoonoses. Cases of infected people involved with crocodile handling and egg collection suggest that the reptile can play an important role in the transmission of leptospirosis (Prerez-Flores et al., 2016). ...
Article
Full-text available
For decades, saltwater crocodiles are feared by the public, but the sentiment has gradually changed because local people livelihood has improved by ecotourism industry. Wild crocodile sighting is now being offered as one of ecotourism products in Sarawak but the activity is based solely on local knowledge. The objective of this study is to determine relative density and distribution of saltwater crocodiles along the Bako River, Sarawak during different monsoon seasons. Standard night spotting technique was deployed during northeast monsoon (NEM), southwest monsoon (SWM) and inter-monsoon (IM). Other works involved documenting riparian landscape along the river and measuring water pH, temperature and salinity. Approximately 117, 60, 92 wild crocodiles had been spotted during SWM, IM, and NEM, respectively. Relative density fluctuated with 3.65, 1.93 and 4.67 non-hatchling/km among seasons. Adults could be seen either resting on river banks or in the middle part of the river while juveniles appeared in small groups near mangrove patches. Data obtained will help relevant state agencies and ecotourism industry players to improve crocodile watching activity offered to tourists. This is important in order to ensure maximum enjoyable experience (without compromising safety) among tourists as well as benefiting local communities. © 2018, Malaysian Society of Applied Biology. All rights reserved.
Book
Leptospira is a Gram-negative bacterium that causes leptospirosis, one of the most important re-emerging zoonotic diseases. The disease is worldwide diffused, and animals are involved in its spreading. Among animals, wildlife play an important role in the epidemiology of leptospirosis, as reservoir of specific Leptospira serovar. Several species are known as Leptospira maintenance host, but other are less investigated and could represent a “new” host involved in its epidemiology. The book “Leptospira Infection in Wild Animals” contains descriptions of leptospirosis epidemiology in several wild animal species, highlighting the infection in different part of world, the most detected Leptospira serovar and the risks of infection for both humans and domestic animals. Data on marine mammals, wild boar, rodent, lagomorph, wild ruminants, amphibian and reptiles, bats and non-human primates Leptospira infection were deeply analysed and discussed in order to better understand their role in the leptospirosis epidemiology.
Article
Full-text available
We describe anti-Leptospira spp. agglutinin in yellow-spotted river turtles (Podocnemis unifilis)in the Amazon region. Ninety-eight serum samples from individuals housed at the Bosque Rodrigues Alves Zoobotanical Garden of Amazonia, Belém, PA, Brazil, were subject to the microscopic agglutination test (MAT) using 19 different Leptospira spp. antigen serogroups. Thirty-four of the 98 samples (35%) were reactive, with titers ranging from 100 to 3200, and eight 8 reactive samples (23.5%) co-agglutinated under two or more serovars.The most common serogroup was Hebdomadis (26.9%, 7/26), followed by Semaranga (23%, 6/26), Shermani (19.2%, 5/26), Djasiman (11.5%, 3/26), and Australis (7.7%, 2/26); Bataviae, Javanica, and Sejroewere represented by a single sample each (3.9%). The presence of turtles reactive to anti-Leptospira spp. antibodies implies exposure to the pathogen.
Article
Full-text available
Bacterial cultures and chemical analyses were performed from cloacal and oral swabs taken from 43 American crocodiles Crocodylus acutus and 28 Morelet’s crocodiles C. moreletii captured in Quintana Roo State, Mexico. We recovered 47 bacterial species (28 genera and 14 families) from all samples with 51.1% of these belonging to the family Enterobacteriaceae. Fourteen species (29.8%) were detected in both crocodile species and 18 (38.3%) and 15 (31.9%) species were only detected in American and Morelet’s crocodiles, respectively. We recovered 35 bacterial species from all oral samples, of which 9 (25.8%) were detected in both crocodile species. From all cloacal samples, we recovered 21 bacterial species, of which 8 (38.1%) were detected in both crocodile species. The most commonly isolated bacteria in cloacal samples were Aeromonas hydrophila and Escherichia coli, whereas in oral samples the most common bacteria were A. hydrophila and Arcanobacterium pyogenes. The bacteria isolated represent a potential threat to crocodile health during conditions of stress and a threat to human health through crocodile bites, crocodile meat consumption or carrying out activities in crocodile habitat. We especially warn about the presence of Salmonella arizonae and S. typhi, which cause enteritis and septicemia in crocodiles and salmonellosis and typhoid fever in humans. The risk of bacterial contamination from crocodiles to humans could increase in the future because of the accelerated destruction of crocodile habitat, which could lead to an augmentation of human−crocodile interactions. Informa- tion on bacterial diversity reported here could help in the choice of antibacterial products in case of infections that are of crocodile origin.
Article
Full-text available
Serum samples from 130 individuals representing 42 species of carnivores, ungulates, and primates from a population of captive mammals in Metropolitan Region in Chile were tested for antibodies against nine serovars of Leptospira interrogans using the microscopic agglutination test. Ten percent of the animals were seropositive to one or more serovars. Seroprevalence was significantly higher in ungulates (20.4%) compared to carnivores (3.8%) and primates (3.4%). There were no significant differences in seroprevalence among sex and age ranges. The most frequent serovar detected was Autumnalis, present in 53.4% of antibody-positive animals. Most positive animals had titers of ≤1 : 200, except for a maned wolf ( Chrysocyon brachyurus ) with titers of 1 : 400 against serovar Hardjo. To the authors' knowledge, this is the first report of Leptospira exposure detected in native endangered pudu ( Pudu puda ) and the first confirmation of exposure to L. interrogans in captive wild mammals in Chile. Leptospirosis should be considered as a differential diagnosis in future disease presentation for hepatitis or abortions in captive mammals in Chile.
Article
Full-text available
Background Leptospirosis is widespread in Mexico, yet the potential distribution and risk of the disease remain unknown. Methodology/Principal Findings We analysed morbidity and mortality according to age and gender based on three sources of data reported by the Ministry of Health and the National Institute of Geography and Statics of Mexico, for the decade 2000 2010. A total of 1,547 cases were reported in 27 states, the majority of which were registered during the rainy season, and the most affected age group was 25–44 years old. Although leptospirosis has been reported as an occupational disease of males, analysis of morbidity in Mexico showed no male preference. A total number of 198 deaths were registered in 21 states, mainly in urban settings. Mortality was higher in males (61.1%) as compared to females (38.9%), and the case fatality ratio was also increased in males. The overall case fatality ratio in Mexico was elevated (12.8%), as compared to other countries. We additionally determined the potential disease distribution by examining the spatial epidemiology combined with spatial modeling using ecological niche modeling techniques. We identified regions where leptospirosis could be present and created a potential distribution map using bioclimatic variables derived from temperature and precipitation. Our data show that the distribution of the cases was more related to temperature (75%) than to precipitation variables. Ecological niche modeling showed predictive areas that were widely distributed in central and southern Mexico, excluding areas characterized by extreme climates. Conclusions/Significance In conclusion, an epidemiological surveillance of leptospirosis is recommended in Mexico, since 55.7% of the country has environmental conditions fulfilling the criteria that favor the presence of the disease.
Article
Full-text available
Leptospirosis is an important public health threat in sub-Saharan Africa but little is known regarding the host spectrum and epidemiology of this zoonotic disease. 289 kidney samples from 69 wild, domestic and peri-domestic species in northern Botswana were screened for the presence of Leptospira sp. Renal carriage was widespread among mammals (31.4%, n=11/35 species), birds (27.8%, n=5/18 species) and reptiles (6.3%, n=1/16 species), including several novel species. Leptospiral surveillance is often limited to the usual suspects: rodents and domestic animals. We identify Leptospira in a wide range of African wildlife, suggesting that leptospirosis transmission and persistence may also involve hosts not normally considered. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Article
Full-text available
In the past decade, leptospirosis has emerged as a globally important infectious disease. It occurs in urban environments of industrialised and developing countries, as well as in rural regions worldwide. Mortality remains significant, related both to delays in diagnosis due to lack of infrastructure and adequate clinical suspicion, and to other poorly understood reasons that may include inherent pathogenicity of some leptospiral strains or genetically determined host immunopathological responses. Pulmonary haemorrhage is recognised increasingly as a major, often lethal, manifestation of leptospirosis, the pathogenesis of which remains unclear. The completion of the genome sequence of Leptospira interrogans serovar lai, and other continuing leptospiral genome sequencing projects, promise to guide future work on the disease. Mainstays of treatment are still tetracyclines and beta-lactam/cephalosporins. No vaccine is available. Prevention is largely dependent on sanitation measures that may be difficult to implement, especially in developing countries.
Article
Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums.
Article
A total of 84 amphibians and reptiles were collected in southern Illinois and cultured for leptospires. All cultures were negative. Sera from 182 specimens were tested, and leptospiral agglutinins were detected in 6 of the 12 species examined. Sera from 18 (26%) of 69 seropositive turtles reacted to Leptospira ballum and 59 (86%) reacted to L. hyos. Inversely, 6 of 9 seropositive snake sera (67%) reacted to L. ballum, but only 1 (11%) reacted to L. hyos. Agglutinins were also detected for L. canicola, L. icterohaemorrhagiae, L. pomona, L. sejroe and L. hardjo. The highest percentage (89.1%) of reactors was in red-eared turtles (Pseudemys scripta elegans). There was no diference in the response of either sex or size classes of the red-eared turtles, although no small turtles were collected. It was postulated that high titers and high reactor rates developed in aquatic turtles in response to continue exposure to water-borne leptospires. In terrestial snakes the mode of infection was probably associated with preying on infected rodents. A total of 84 amphibians and reptiles were collected in southern Illinois and cultured for leptospires. All cultures were negative. Sera from 182 specimens were tested, and leptospiral agglutinins were detected in 6 of the 12 species examined. Sera from 18 (26%) of 69 seropositive turtles reacted to Leptospira ballum and 59 (86%) reacted to L. hyos. Inversely, 6 of 9 seropositive snake sera (67%) reacted to L. ballum, but only 1 (11%) reacted to L. hyos. Agglutinins were also detected for L. canicola, L. icterohaemorrhagiae, L. pomona, L. sejroe and L. hardjo. The highest percentage (89.1%) of reactors was in red-eared turtles (Pseudemys scripta elegans). There was no diference in the response of either sex or size classes of the red-eared turtles, although no small turtles were collected. It was postulated that high titers and high reactor rates developed in aquatic turtles in response to continue exposure to water-borne leptospires. In terrestial snakes the mode of infection was probably associated with preying on infected rodents.
Article
Antibacterial agents were purified from Siamese crocodile serum by anion exchange, gel filtration and reversed phase HPLC. Six antibacterial agents designed as Hp14, Hp15, Hp17, Hp31, Hp36 and Hp51 were purified and proved to carry activity against Salmonella typhi, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Vibrio chorelae. The mass analysis of MALDI-TOF for antibacterial agent of Hp14, Hp15 and Hp51 revealed that they are small molecule with a molecular mass less than 1 kDa. The scanning electron microscopy demonstrated that these agents targeted the bacterial membrane and they act like as antimicrobial peptides. The antibacterial agent in the serum may represent the first line of an immune system in a freshwater crocodile.